Low-temperature inducible expression vector

Abstract

A vector which is characterized in containing each of the following elements: (1) a promoter which shows its action in the host to be used; (2) regulatory region for regulating the action of the promoter of (1); and (3) a region which codes for the 5′-untranslated region derived from cold-shock protein gene mRNA or a region which codes for the region where substitution, deletion, insertion or addition of at least one base is applied to the said untranslated region.

1. A vector which is characterized in containing each of the following elements:
 (1) a promoter which is derived from cold-shock protein gene, and which shows its action in the host to be used;  
 (2) regulatory region for regulating the action of the promoter of (1), wherein said regulatory region is located downstream of the promoter of (1); and  
 (3) a region which codes for the 5′-untranslated region derived from cold-shock protein gene mRNA or a region which codes for the region where substitution, deletion, insertion, or addition of at least one base is applied to said untranslated region.  
 
     
     
       2. A vector according to  claim 1  in which a promoter derived from cold-shock protein gene is a promoter derived from cspA gene of  E. coli.    
     
     
       3. A vector according to  claim 2  in which a promoter derived form cspA gene of  E. coli  is a promoter containing a base sequence as shown in SEQ ID NO: 5 in the Sequence Listing. 
     
     
       4. A vector according to  claim 1  in which regulatory region is an operator. 
     
     
       5. A vector according to  claim 4  in which an operator is a lac operator. 
     
     
       6. A vector according to  claim 5 , which contains a region coding for the 5′-untranslated region containing lac operator, wherein a base sequence of said region is selected from the group consisting of the base sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 in the Sequence Listing. 
     
     
       7. A vector according to  claim 1  which contains a region which codes for the 5′-untranslated region derived from cspA gene mRNA or  E. coil  or a region which codes for the region where substitution, deletion, insertion, or addition of at least one base is applied to said untranslated region. 
     
     
       8. A vector according to  claim 7  which contains a region coding for the 5′-untranslated region, said region comprising nucleotides 483-516 and 547-620 of the cspA gene represented by SEQ ID NO: 6 in the Sequence Listing. 
     
     
       9. A vector according to  claim 7  which contains a region coding for the 5′untranslated region containing the region of +27 to +55 and +86 to +159 counted from the initiation point for transcription of the cspA gene, wherein said initiation point for translation is nucleotide 462 in the cspA gene represented by SEQ ID No:6 in the Sequence Listing. 
     
     
       10. A vector according to  claim 1  which further contains, in the downstream of the 5′-untranslated region, a base sequence having a complementarity to the anti-downstream box sequence of ribosomal RNA of the host used. 
     
     
       11. A vector according to  claim 10  which contains, in the downstream of the 5′-untranslated region, a base sequence as shown in SEQ ID NO: 28 in the Sequence Listing. 
     
     
       12. A method for the expression of the desired protein which is characterized in containing the following steps:
 1) a step where a host is transformed with a vector containing: 
 a) a promoter which is derived from cold-shock protein gene and which shows its action in the host to be used;  
 b) a regulatory region for regulating the action of the promoter of a), wherein said regulatory region; and  
 c) a region which codes for the 5′-untranslated region derived from cold-shock protein gene mRNA or a region which codes for the region where substitution, deletion, insertion, or addition of at least one base is applied to the untranslated region wherein a gene coding for the desired protein is integrated;  
 
 2) a step where the resulting transformant is incubated; and  
 3) a step where action of promoter is induced via a function of a regulatory region and, at the same time, incubating temperature is made lower than the ordinary temperature to express the desired protein.